Phenotypic Scoring of Zebrafish Larvae

The zebrafish larvae were visually evaluated at 5 dpf using a fluorescence microscope (Fluorescence Stereo Microscope M205 FA, equipped with DFC490 camera, Leica, Mannheim, Germany). This study was non-blinded. Mortalities were noted as described above and surviving larvae were scored according to phenotypic abnormalities. Phenotypic scoring was based on the presence of abnormal pigmentation, presence of swelling of the region around the heart for pericardial oedema, presence of a dense red region in the head for cerebral haemorrhage, tail kinking for tail malformation, and angle deviation of the lower jaw compared to the larvae in negative control for jaw malformation [6 (link)]. Each larva was scored individually from (0) healthy, (1) mild, (2) moderate, and (3) severe malformations in every phenotype. Deceased larvae were excluded from the scoring system.